In the natural environment, bacteria grow predominantly within sessile, matrix-enclosed communities known as biofilms, rather than as unattached planktonic cells. Biofilms protect resident bacteria from attack by the immune system, compromise antimicrobial therapy, and disperse planktonic cells, which promote the spread of infection to distant body sites. Biofilms complicate about 65 percent of recalcitrant bacterial infections. Nevertheless, the regulatory mechanisms of biofilm development, especially biofilm dispersal, remain poorly defined in any species. Our studies revealed that the RNA-binding global regulatory protein CsrA is a potent repressor of biofilm formation in Escherichia coli K-12 and pathogenic relatives. This effect is mediated primarily through its regulation of intracelluar glycogen synthesis and turnover. Further evidence supports a role for glycogen in the synthesis of a polysaccharide adhesin. Mutations that inhibit biofilm formation were isolated in a 4-gene operon, which was cloned and found to needed for the production of a GIcN-rich polysaccharide. Remarkably, csrA-induction within cells of a preformed biofilm caused extensive dispersal, releasing viable planktonic cells. This finding offers a key to unlock the biochemical and genetic bases of biofilm dispersal. The Aims of this proposal are to: 1) Characterize a novel polysaccharide adhesin needed for E. coli biofilm formation. The molecular genetics of its synthesis, its chemistry, and its function in biofilm formation will be assessed. 2) Investigate the genetic and metabolic determinants of polysaccharide production. The precursor-product relationship of glycogen to the polysaccharide adhesin will be examined by ELISA analysis of existing mutants and by 13C NMR labeling studies in conjunction with appropriate structural and regulatory mutations. The genetic regulation of its biosynthesis will be examined with reporter fusions and other approaches. 3) Biofilm dispersal will be assessed systematically by examining the effects of csrA induction on the polysaccharide adhesin, membrane and periplasmic proteins, LPS and the transcriptome. Results will pave the way for studies defining the mechanisms of the dispersal process.The long-range goal of these studies is to develop a full understanding of the regulatory factors, metabolic pathways and structural elements that interact in biofilm formation, and thereby obtain useful information for combating biofilm infections.